Project Summary: The specification of three cone photoreceptor subtypes enables high-acuity daytime and trichromatic color vision in humans. During terminal differentiation, cone cells express opsins that define their functionalities as blue (S-opsin), green (M-opsin), or red (L-opsin) light detectors. How the three cone subtypes are generated in humans remains largely unknown. Elucidating these mechanisms will be critical for understanding and treating visual disorders including macular degeneration, cone dystrophy, retinitis pigmentosa, foveal hypoplasia, and color blindness. The goal of this project is to determine the mechanisms controlling the specification of S, M, and L cones in the human retina. Cone subtype specification occurs in a two-step process. First, a decision occurs between S and L/M cone fates. If the L/M fate is chosen, a subsequent choice is made between L and M cone fates. To study these processes, we genetically and pharmacologically manipulated human retinal organoids to test mechanisms of retinal development. We found that dynamic expression of thyroid hormone (TH)-regulating genes within the retina ensures low TH signaling early to specify S cones and high TH signaling late to produce L/M cones. Our work established human retinal organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair (Eldred et al., 2018, Science). We found that S, M, and L cones are generated in a temporal progression during development. How temporal and spatial inputs are integrated in the human retina to generate patterns of cones is poorly understood. We developed dissection, imaging, and cell identification approaches to assess the spatial distributions of cone subtypes and generate the first maps of cone subtypes across intact human retinas (Aim 1). We found that temporal regulation of TH signaling is critical for establishment and maintenance of cone subtype fates. To evaluate thyroid dysregulation and cone fate plasticity, we will define the temporal windows of TH regulation in organoids and examine cone fates in retinas from patients with late-onset thyroid dysregulation. We will determine the roles of TH-regulating genes in cone subtype specification in organoids (Aim 2). Additionally, we found that retinoic acid (RA) signaling induces S cone fate and suppresses L/M cone fate early in decision 1, and induces L cone fate and suppresses M cone fate late in decision 2. We will characterize temporal windows of RA action and assess the function and expression of RA-regulating genes in the specification of cone subtype fates in organoids (Aim 3). These experiments will delineate how regulation of TH and RA signaling specify cone subtypes in the human retina. Moreover, these experiments will provide the first direct mechanistic insight into L and M cone specification with important implications for understanding red/green color blindness and developing regenerative therapeutics for macular degeneration.